Cell Lysis and purification of inclusion bodies

6. Resuspend pellet in 10ml of washing buffer and transfer to a Nalgene centrifuge tube (suitable for the Sorvall SS-34 rotor)
7. Do three freeze-thaw cycles: freeze in dry-ice and ethanol (~10 minutes), thaw in the 37 C waterbath (~ 10 minutes).
8. Fill Nalgene tube up to approximately two-thirds with washing buffer and then sonicate. Use the large tip. Program for 10 s on and 10 s off, for 2 minutes total process time. Use temperature control probe set to 37 C
9. Spin down cells in Sorvall SS-34 rotor for 15 minutes at 12,000 rpm
10. Remove supernatant and resuspend pellet in 10ml washing buffer
11. Fill Nalgene up to approximately two-thirds with washing buffer and then sonicate as before.
12. Repeat centrifugation as in step 9.

Protein refolding

16. Add the filtered solubilization mixture to 2L of refolding buffer and allow to mix slowly at 4C overnight. Cover the beaker but do not seal.
17. Check for presence of sulfur smell (cysteamine) – it may be necessary to refold for longer if the sulfur smell lingers
18. Use a filtertop filter unit to filter the 2L of refolded protein and transfer to the large concentrator
19. Use a 5kDa MWCO filter (Millipore) – shiny side facing up. Start concentrator, making sure that solution is flowing slowly. Concentrate until volume is approximately 50ml. It may be necessary to turn of the stirbar as the solution goes below 100ml, as the stirbar may generate bubbles.
20. Remove solution from the concentrator and check protein concentration at A280 using the nanodrop (retain a portion of the flow through from the concentration step for a blank).

FPLC

21. Take 500µl of the protein solution and inject into the S75 FPLC column (flow rate should be set at 0.5ml/min.
22. Protein should elute at ~17ml.

Protein Characterization

23. Protein may be further concentrated using a centricon, down to one-tenth of the volume. At this point the buffer may also be exchanged
24. Measure the protein concentration again using the nanodrop
25. Run an aliquot of the protein on SDS-PAGE to verify size and determine purity.